Ação do nitróxido tempol sobre a atividade do complexo enzimático NADPH oxidase (Nox2) em neutrófilos

Abstract

The identification of new targets for controlling inflammatory processes is, almost certainly, the major challenge that hampers the development of new anti-inflammatory drugs. Thus, the modulation of intracellular signaling pathways in phagocytes emerges as an interesting way to achieve this goal. However, this strategy can bring the effect of lowering the host defense against pathogens. This study aimed to examine whether the nitroxide 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempol) has an effect on production of oxidants by inflammatory neutrophils through regulation of protein kinase activity and protein disulfide isomerase (PDI), a chaperone whose active sites containing the motif Cys-Gly-His-Cys (CXXC) and is involved in the assembly of the NADPH oxidase enzyme complex (Nox2) on phagocytes. Different biochemical pathways were activated for the release of reactive oxygen species by inflammatory neutrophils, and, in parallel, the protein kinase activities were determined under Tempol treatment of phagocytes. The nitroxide significantly inhibited oxidative burst of neutrophils in a concentration dependent manner. This effect was detected by oxygen consumption, where the IC50 was found to be 45±4 µM. By means of chemiluminescence luminol- or isoluminol-dependent assay, we showed that Tempol caused a delay in the lag period of neutrophils Nox2 activation under different stimuli. Accordingly, the nitroxide inhibited the activity of protein kinases in neutrophils stimulated by various biochemical pathways, as quantitated by chemiluminescent assays and dot blot test. Under the same conditions, Tempol reduced neutrophil fungicidal activity against Candida albicans. In the presence of Tempol, PDI reductase activity was reversibly affected both in vitro and in stimulated inflammatory neutrophils, whose IC50 of 35±3.3 µM was calculated by fluorescent methodology. This inhibitory activity was confirmed by the spectrophotometric insulin method. Recombinant PDI was used for the purpose of studying the mechanism of inhibition that Tempol exerts on this thiol oxide reductase enzyme, by mass spectrometry. In the analysis of total protein, 1 and 4 molecules of Tempol were detected bound to the protein. However, only one molecule of the nitroxide was found covalently linked to PDI. More specifically, Cys400 has been changed by Tempol. Together, these results reveal a novel mechanism of Tempol on the regulation of enzymes associated with Nox2 complex inflammatory activity of neutrophils, which have potential clinical applications for therapeutic intervention in pathological processes. However, the promising use of Tempol as an anti-inflammatory agent must be taken with caution, since this nitroxide decreased neutrophil microbicidal response.